Microbials for salmonella inhibition

Abstract

The present disclosure relates to microbials for use in E. coli, Salmonella, Enterococcus and / or Clostridium inhibition in animals and their environment. More particularly, the present disclosure relates to isolated Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-I60 (NRRL No. B-68472), and strains having all of the identifying characteristics of these strains, for a use comprising the above-mentioned use.

Description

60421-435175MICROBIALS FOR SALMONELLA INHIBITION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application Serial No. 63 / 742,175, filed on January 6, 2025, the entire disclosure of which is incorporated herein by reference.FIELD OF THE DISCLOSURE

[0002] The invention relates to microbials for use in inhibiting the growth and or reproduction of pathogenic bacteria, including E. coli, Clostridium, Enterococcus, and Salmonella.BACKGROUND

[0003] Salmonella is one of the most widespread foodborne pathogens worldwide, and Salmonella infections in humans represent a large global health burden. Globally, non- typhoidal Salmonella is responsible for approximately 93 million cases of gastroenteritis and 155,000 fatalities annually. The main source of Salmonella infections in humans is contaminated animal-derived foodstuffs, with pork and poultry products being some of the most important sources. Beyond the health risks to humans, salmonellosis causes economic losses as well. It is estimated that foodborne salmonellosis costs the U.S. $2.7 billion per year.

[0004] Controlling Salmonella and other known pathogenic bacteria requires a combination of different pre -harvest and post-harvest methods such as biosecurity, cleaning and disinfection procedures, feed additives, antibiotics, vaccines, monitoring, and much more. Management of Salmonella is difficult because animals can serve as asymptomatic carriers spreading the pathogen through feces, environmental contamination, and food products. It is reported that Salmonella can persist in the environment for several months to years showing the importance of using methods to reduce the bioburden in the environment and the gastrointestinal tract of animals. Mitigation strategies must cover the entire production chain from start to finish as Salmonella can be present and cause contamination at every stage.

[0005] Regardless of industry segments, producers face universal pressure to implement technologies to lower the Salmonella burden starting in the live production phase. Control in poultry, ruminant, and pig production is a complex and ongoing challenge for producers. According to the Kauffman-White Scheme for classification, there are over 2,500 serovars of Salmonella making it a difficult bacterial species to target. Beyond the continuously changing60421-435175and increasing numbers of serovars; Salmonella is difficult to control due to its mechanisms to co-evolve with its host by altering cellular processing that favor bacterial survival and intracellular proliferation. Salmonella spp. are also capable of forming biofilms that are resistant to antimicrobials and contribute to the increased virulence of the bacteria establishing a chronic infection.

[0006] The control of this pathogen is critical for animal welfare, food safety, and consumer trust. Therefore, novel technologies to help manage or reduce levels of Salmonella within animal production systems are crucial. Applicants have developed a microbial composition that reduces the growth of Salmonella. The microbial compositions described herein offer a commercial benefit by providing these properties, or a combination thereof, in a single microbial composition.SUMMARY

[0007] The present invention relates to microbial compositions and methods for inhibiting pathogenic bacteria in an animal, in domesticated animal enclosures, and in agriculture and livestock products. In one embodiment the compositions are formulated as direct fed compositions for administration to animals for inhibiting the growth and replication of Salmonella in animals. More particularly, the invention relates to isolated Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG- UC25 (NRRL No. B-68471) and MDG-160 (NRRL No. B-68472), and strains having all of the identifying characteristics of these strains, for a use in inhibiting the growth and / or replication of a pathogenic bacteria selected from E. coll, Clostridium, Enterococcus, and Salmonella. In one embodiment compositions are provided comprising one or more Bacillus strains selected from the group consisting of MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471) and MDG-160 (NRRL No. B- 68472), that are formulated for ingestion by animals. In one embodiment the compositions are formulated as a dry powder or pellet. In one embodiment the composition comprises a pharmaceutically acceptable ingredient selected from the group consisting of gelatin, cellulose, starch, excipient, binder, anti-caking agent, preservative, or combination thereof, wherein the Bacillus strain is stabilized by the pharmaceutically acceptable ingredient such that the Bacillus strain is able to survive, reproduce, and grow in the digestive tract of the animal.

[0008] An animal’s gastrointestinal tract is constantly challenged by large numbers of bacteria, viruses, fungi, and protozoa found in feed, bedding, and the environment. The gastrointestinal tract has a sophisticated system to counter these potential pathogens60421-435175consisting of physical, chemical, and immunological lines of defense. Beneficial bacteria are an important part of this system because they provide animals with bacteria that assist in establishment (or reestablishment) of a normal bacterial profile, they strengthen the animal’s immune system, and they help to fight disease (e.g., disease caused by Salmonella in animals). Due to the importance of preventing and treating Salmonella associated disease in animals, both to the agricultural industry, and to the human food supply, direct-fed microbial strains are needed that inhibit Salmonella in animals, such as agricultural animals.

[0009] Applicants have developed direct-fed microbials that result in inhibition of pathogenic bacteria, including the inhibition of E. coll, Clostridium, Enterococcus and Salmonella. The direct-fed microbials and compositions comprising the direct-fed microbials described herein offer a commercial benefit by providing E. coli, Clostridium, Enterococcus, and Salmonella inhibition in animals, such as agricultural animals. In addition, the direct-fed microbial compositions described herein result in a reduction or elimination of the use of antibiotics which reduces the overall cost of animal feed and reduces the development of antibiotic resistance.

[0010] Methods and compositions are provided for inhibiting E. coli, Clostridium, Enterococcus, and Salmonella in animals, and in the animal’s local environment, or in agricultural fields and their associated crops. In various embodiments, the animal can be selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal. In the embodiment where the animal is a poultry species, and the poultry species can be selected from the group consisting of a broiler, a chicken, a layer, a breeder, a turkey, a turkey poult, a gosling, a duckling, a guineakeet, a pullet, a hen, a rooster, a cockerel, and a capon. In the embodiment where the animal is a porcine species, the porcine species can be selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig. In addition, the number of pathogenic E. coli, Clostridium, Enterococcus, and Salmonella present in an animals local environment, including the animal’s enclosure, bedding, feed, manure, can be reduced by contacting the surfaces of the animals local environment with a compositions comprising isolated Bacillus strains MDG-844 (NRRL No. B-68469), MDG- UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B- 68472), or any combination thereof, and strains having all of the identifying characteristics of these strains. In one embodiment a composition comprising one or more Bacillus strains selected from Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B- 68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), and any combination thereof, is provided. In one embodiment the composition is used to inhibit60421-435175growth of Salmonella isolates, enhance Salmonella antimicrobial susceptibility, inhibit Salmonella biofilm formation, and / or inhibit Salmonella adhesion and invasion of target host cells.

[0011] In one embodiment the Bacillus comprising compositions disclosed herein are used to inhibit growth of Salmonella isolates, enhance Salmonella antimicrobial susceptibility, inhibit Salmonella biofilm formation, and / or reduce Salmonella adhesion and invasion of target host cells. In one embodiment the compositions comprise one or more Bacillus strains selected form the group consisting of Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472) or a cell lysate or a cell-free fermentate of one or more of said Bacillus strains.

[0012] In one embodiment, a method of feeding an animal a composition that improves animal health is provided. The method comprises the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of isolated Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof, wherein the Bacillus strain causes Salmonella inhibition in the animal.

[0013] In another embodiment, a method of controlling a detrimental effect of a pathogenic bacteria, including for example E. coli, Clostridium, Enterococcus or Salmonella pathogenic bacteria is provided. The method comprises the steps of administering to an animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of isolated Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-60421-43517568472), and combinations thereof, and controlling the detrimental effect of the pathogenic bacteria in the animal.

[0014] In yet another embodiment, a commercial package is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain selected from the group consisting of isolated Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof. In one embodiment the commercial package is formulated as a powder or liquid. In another embodiment, the commercial package is formulated with a fertilizer. In an additional embodiment the commercial package is formulated as a pellet, granules, capsules, or tablet. In one embodiment the commercial package is formulated with a pharmaceutically acceptable carrier for ingestion by an animal wherein the pharmaceutically acceptable carrier is a natural or synthetic polymer, including for example, pectin, chitosan, guar gum, chondroitin sulfate, dextran, cyclodextrin, inulin, xanthan gum, amylose, alginates, shellac, locust bean gum and any combination thereof. In another embodiment the commercial package comprises a binder, present at about 2-10% of the total weight of the commercial product. In one embodiment the binder comprises one or more compounds selected from the group consisting of clay, yeast cell wall components, aluminum silicate, glucan, or other known binders.

[0015] In still another embodiment, a feed additive for an animal feed is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain selected from the group consisting of isolated Bacillus strains MDG-844 (NRRL No. B- 68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof.

[0016] In another illustrative embodiment, an additive for the drinking water of an animal is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain selected from the group consisting of isolated MDG-844 (NRRL No. B-60421-43517568469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof.

[0017] In another aspect, an animal feed composition is provided comprising one or more nutrient compounds selected from the group consisting of lipids, protein, fiber, carbohydrate, minerals and vitamins; and an isolated Bacillus strain selected from the group consisting of Bacillus strain selected from the group consisting of isolated Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof. In one embodiment the feed composition comprises a pellet comprising nutrient compounds, wherein the pellet is coated or admixed with one or more of the Bacillus strains selected from the group consisting of isolated Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472).

[0018] In yet another embodiment, a method of feeding an animal is provided. The method comprises the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of isolated Bacillus strains MDG-844 (NRRL No. B- 68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof.

[0019] In another illustrative aspect, a method of treating an animal to inhibit a bacterial disease caused by Salmonella is provided. The method comprises feeding the animal a60421-435175composition comprising an effective amount of an isolated Bacillus strain selected from the group consisting of Bacillus strain selected from the group consisting of isolated Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof, and inhibiting Salmonella in the animal.

[0020] In an additional aspect, a method of inhibiting the growth and / or reproduction of pathogenic bacteria on a surface is provided. The method comprises inhibiting pathogenic bacteria, optionally wherein the pathogenic bacteria are selected from E. coli, Salmonella, Enterococcus and / or Clostridium pathogens, by contacting said pathogenic bacteria with a composition comprising an isolated Bacillus strain, selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG- UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain MDG-160 (NRRL No. B-68472), and combinations thereof or a lysate, or a fermentation product of said Bacillus strain. In one embodiment, the surface to be treated is a surface on an article selected from the group consisting of animal bedding, animal bedding shavings, animal litter, manure, domesticated animal enclosures, crop plants, crop fields, produce, and meat products. In one embodiment, the composition is in the form of a powder. In one embodiment, the composition is in the form of a liquid and the pathogenic bacteria is contacted with said composition via a spray, dip, or wash.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Fig. 1 In vitro Salmonella colonization of C. elegans by treatment. Salmonella colonization is classified by CFU per worm (Logic).

[0022] Fig. 2 is a graph presenting data showing the effect of cell-free fermentates from Bacillus strain 844 (NRRL No. B-68469) and Bacillus strain UF7 (NRRL No. B-68470) on biofilm growth of a poultry origin Salmonella isolate.60421-435175

[0023] Fig. 3 is a graph presenting data showing a reduction in Salmonella invA gene quantities by more than 1 log when Salmonella Enteritidis was incubated with Bacillus strain 844 (NRRL No. B-68469), Bacillus strain UC25 (NRRL No. B-68471). or Bacillus strain UF7 (NRRL No. B-68470) by compared to the control (p<0.001).

[0024] Fig. 4 is a graph presenting data showing a reduction in attached or invaded Salmonella of the swine intestinal epithelial cells of the IPEC-J2 line (p<0.001) when Salmonella Enteritidis was incubated with Bacillus strain 844 (NRRL No. B-68469).DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0025] Methods and compositions are provided for inhibiting E. coli, Salmonella, Enterococcus and / or Clostridium in animals. In various embodiments, the animal can be selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal. In the embodiment where the animal is a poultry species, the poultry species can be selected from the group consisting of a broiler, a chicken, a layer, a breeder, a turkey, a turkey poult, a gosling, a duckling, a guineakeet, a pullet, a hen, a rooster, a cockerel, and a capon. In the embodiment where the animal is a porcine species, the porcine species can be selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.

[0026] In one embodiment, a method of feeding an animal to prevent or reduce the presence of a pathogenic bacteria such as Salmonella is provided. The method comprises the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B- 68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B- 68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof, wherein the Bacillus strain causes E. coli, Salmonella, Enterococcus and / or Clostridium inhibition in the animal.

[0027] In another embodiment, a method of controlling a detrimental effect of E. coli, Salmonella, Enterococcus, and / or Clostridium is provided. The method comprises the steps of administering to an animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-60421-43517568470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B- 68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof, and controlling the detrimental effect of E. coli, Salmonella, Enterococcus and / or Clostridium in the animal. In one embodiment, a method of controlling a detrimental effect of E. coli, Salmonella, Enterococcus and / or Clostridium is provided wherein the method comprises the steps of administering to an animal a feed composition or drinking water that comprises an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B- 68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), and any combinations thereof.

[0028] In yet another embodiment, a commercial package is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof.

[0029] In still another embodiment, a feed additive for an animal feed is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof. In one embodiment, a feed additive for an animal feed is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), and combinations thereof.60421-435175

[0030] In another illustrative embodiment, an additive for the drinking water of an animal is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG- UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof. In one embodiment, an additive for the drinking water of an animal is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), and combinations thereof.

[0031] In another aspect, an animal feed composition is provided comprising a nutrient formulation and an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof. In one embodiment, an animal feed composition is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), and combinations thereof.

[0032] In yet another embodiment, a method of feeding an animal is provided. The method comprises the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG- UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B- 68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof.60421-435175

[0033] In an additional embodiment, a method of inhibiting pathogenic bacteria, optionally a Salmonella strain, on a surface is provided. The method comprises contacting pathogenic bacteria, optionally wherein the pathogenic bacteria are selected from E. coll, Salmonella, Enterococcus and / or Clostridium pathogens with an effective amount of a composition comprising an isolated Bacillus strain, selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG- UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain MDG-160 (NRRL No. B-68472), and combinations thereof, or a lysate, or a fermentation product of said Bacillus strain. The surface to be treated can be any surface known to harbor pathogenic E. coli, Salmonella, Enterococcus and / or Clostridium strains. Such surfaces include the surfaces of crops, agricultural fields, produce and meat products as well as surfaces in contact with animals, including for example animal bedding, animal enclosures, and surfaces exposed to human or animal waste. In one embodiment the surfaces are contacted by spraying a liquid composition comprising the Bacillus strains disclosed herein onto the surface. In one embodiment the surfaces are contacted by washing the surface with a liquid composition comprising the Bacillus strains disclosed herein onto the surface. In one embodiment the surface is contacted with a lysate, or a fermentation product of one or more of the Bacillus strains disclosed herein. In one embodiment the surface is contacted with a powder form of the Bacillus compositions as disclosed herein.

[0034] Any suitable method known in the art can be used for contacting the surface to be treated with an effective amounts of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG- 844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain MDG-160 (NRRL No. B-68472), and combinations thereof or a lysate, or a fermentation product of said Bacillus strain, or combinations thereof.

[0035] In an embodiment, the surface to be treating is a surface on an article selected from the group consisting of animal bedding, animal bedding shavings, animal litter, manure, domesticated animal enclosures, crop plants, crop fields, produce, and meat products. In an60421-435175embodiment, the composition is in the form of a powder. In another embodiment, the composition is formulated as a liquid and the pathogenic bacteria is contacted with said composition via a spray, dip, or wash.

[0036] The following clauses, and combinations thereof, provide various additional illustrative aspects of the invention described herein. The various embodiments described in this section titled “DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS” are applicable to any of the following embodiments of the invention described in the numbered clauses below.

[0037] 1. A method of feeding an animal, the method comprising the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B- 68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B- 68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof, wherein the Bacillus strain causes E. coli, Salmonella, Enterococcus and / or Clostridium inhibition in the animal.

[0038] 2. The method of clause 1 wherein the animal has a disease caused by E. coli, Salmonella, Enterococcus and / or Clostridium.

[0039] 3. The method of clause 1 wherein the animal is selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal.

[0040] 4. The method of clause 3 wherein the poultry species is a broiler chicken.

[0041] 5. The method of any one of clauses 1 to 4 wherein the E. coli, Salmonella, Enterococcus and / or Clostridium inhibition prevents E. coli, Salmonella, Enterococcus and / or Clostridium disease in the animal.

[0042] 6. The method of any one of clauses 1 to 4 wherein the E. coli, Salmonella, Enterococcus and / or Clostridium inhibition reduces E. coli, Salmonella, Enterococcus and / or Clostridium, disease in the animal.

[0043] 7. The method of clause 3 wherein the animal is a porcine species and the porcine species is selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.60421-435175

[0044] 8. The method of any one of clauses 1 to 7 wherein the Bacillus strain produces an enzyme selected from the group consisting of an a-galactosidase, a protease, a lipase, an amylase, a xylanase, a cellulase, and combinations thereof.

[0045] 9. The method of any one of clauses 1 to 8 further comprising the step of administering to the animal another bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[0046] 10. The method of any one of clauses 1 to 9 wherein the strain administered is Bacillus strain MDG-844 (NRRL No. B-68469), or MDG-UF7 (NRRL No. B-68470).

[0047] 11. The method of any one of clauses 1 to 9 wherein two or more strains are administered, wherein the two or more strains are selected from Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470). MDG-UC25 (NRRL No. B-68471) and MDG-160 (NRRL No. B-68472).

[0048] 12. The method of any one of clauses 1 to 9 wherein three or more strains are administered, wherein the three or more strains are selected from Bacillus strains MDG- 844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B- 68471) and MDG-160 (NRRL No. B-68472). .

[0049] 13. The method of any one of clauses 1 to 9 wherein the strain administered is Bacillus strain MDG-UF7 (NRRL No. B-68470), optionally in combination with one or more strains selected from MDG-844 (NRRL No. B-68469), MDG-UC25 (NRRL No. B- 68471), and MDG-160 (NRRL No. B-68472) ).

[0050] 14. The method of any one of clauses 1 to 9 wherein Bacillus strains MDG- 844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B- 68471), and MDG-160 (NRRL No. B-68472) are all administered.

[0051] 15. The method of any one of clauses 1 to 14 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 103CFU / gram of the feed composition to about 5.0 x 1012CFU / gram of the feed composition.

[0052] 16. The method of any one of clauses 1 to 14 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 103CFU / gram of the feed composition to about 1.0 x 107CFU / gram of the feed composition.

[0053] 17. The method of any one of clauses 1 to 14 wherein the Bacillus strain is administered in the feed composition at a dose greater than about 7.0 x 104CFU / gram of the feed composition.

[0054] 18. The method of any one of clauses 1 to 17 further comprising the step of administering an antibiotic to the animal.60421-435175

[0055] 19. The method of any one of clauses 1 to 18 further comprising the step of administering to the animal an enzyme selected from the group consisting of a galactosidase, a protease, a lipase, an amylase, a hemicellulase, an arabinoxylanase, a xylanase, a cellulase, an NSPase, a phytase, and combinations thereof.

[0056] 20. The method of clause 19 wherein the enzyme is an NSPase or a phytase.

[0057] 21. The method of clause 3 wherein the animal is a companion animal.

[0058] 22. The method of clause 21 wherein the animal is a canine species or a feline species.

[0059] 23. The method of clause 3 wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0060] 24. The method of clause 3 wherein the animal is a sow and the Bacillus strain is administered during gestation.

[0061] 25. The method of any one of clauses 1 to 24 wherein the feed composition is administered daily to the animal.

[0062] 26. The method of clause 1 wherein the animal is selected from the group consisting of a chicken, a pig, a horse, a cow, a turkey, a goat, a sheep, a quail, a pheasant, an ostrich, a duck, a fish, a crustacean, and combinations thereof.

[0063] 27. A method of controlling a detrimental effect of E. coli, Salmonella, Enterococcus and / or Clostridium, the method comprising the steps of administering to an animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof, and controlling the detrimental effect of E. coli, Salmonella, and / or Clostridium in the animal.

[0064] 28. The method of clause 27 wherein the animal has a disease caused by E. coli, Salmonella, Enterococcus and / or Clostridium, optionally wherein the Salmonella species is Salmonella enterica.

[0065] 29. The method of clause 27 wherein the animal is selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal.60421-435175

[0066] 30. The method of clause 29 wherein the poultry species is a broiler chicken.

[0067] 31. The method of any one of clauses 27 to 30 wherein the E. coli, Salmonella, Enterococcus and / or Clostridium inhibition prevents E. coli, Salmonella, Enterococcus and / or Clostridium disease in the animal.

[0068] 32. The method of any one of clauses 27 to 30 wherein the E. coli, Salmonella, Enterococcus and / or Clostridium inhibition reduces E. coli, Salmonella, Enterococcus and / or Clostridium disease in the animal.

[0069] 33. The method of clause 29 wherein the animal is a porcine species and the porcine species is selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.

[0070] 34. The method of any one of clauses 28 to 34 wherein the Bacillus strain produces an enzyme selected from the group consisting of an a-galactosidase, a protease, a lipase, an amylase, a xylanase, a cellulase, and combinations thereof.

[0071] 35. The method of any one of clauses 28 to 35 further comprising the step of administering to the animal another bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[0072] 36. The method of any one of clauses 27 to 25 wherein the strain administered is Bacillus strain MDG-844 (NRRL No. B-68469).

[0073] 37. The method of any one of clauses 27 to 35 wherein the strain administered is Bacillus strain MDG-UF7 (NRRL No. B-68470).

[0074] 38. The method of any one of clauses 27 to 35 wherein the strain administered is Bacillus strain MDG-UC25 (NRRL No. B-68471).

[0075] 39. The method of any one of clauses 27 to 35 wherein the strain administered is Bacillus strain MDG-160 (NRRL No. B-68472).

[0076] 40. The method of any one of clauses 27 to 35 wherein two or more strains are administered, wherein the two or more strains are selected from Bacillus strains MDG- 844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B- 68471) and MDG-160 (NRRL No. B-68472).

[0077] 41. The method of any one of clauses 27 to 35 wherein the strain administered is Bacillus strain MDG-UF7 (NRRL No. B-68470), optionally in combination with one or more strains selected from MDG-844 (NRRL No. B-68469), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472).

[0078] 42. The method of any one of clauses 27 to 35 wherein the strain administered is Bacillus strain MDG-844 (NRRL No. B-68469), optionally in combination60421-435175with one or more strains selected from MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472)

[0079] 43. The method of any one of clauses 27 to 35 wherein Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472) are all administered.

[0080] 44. The method of any one of clauses 27 to 43 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10 CFU / gram of the feed composition to about 5.0 x 1012CFU / gram of the feed composition.

[0081] 45. The method of any one of clauses 27 to 43 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 103CFU / gram of the feed composition to about 1.0 x 107CFU / gram of the feed composition.

[0082] 46. The method of any one of clauses 27 to 43 wherein the Bacillus strain is administered in the feed composition at a dose greater than about 7.0 x 104CFU / gram of the feed composition.

[0083] 47. The method of any one of clauses 27 to 46 further comprising the step of administering an antibiotic to the animal.

[0084] 48. The method of any one of clauses 27 to 47 further comprising the step of administering to the animal an enzyme selected from the group consisting of a galactosidase, a protease, a lipase, an amylase, a hemicellulase, an arabinoxylanase, a xylanase, a cellulase, an NSPase, a phytase, and combinations thereof.

[0085] 49. The method of clause 48 wherein the enzyme is an NSPase or a phytase.

[0086] 50. The method of clause 29 wherein the animal is a companion animal.

[0087] 51. The method of clause 50 wherein the animal is a canine species or a feline species.

[0088] 52. The method of clause 29 wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0089] 53. The method of clause 29 wherein the animal is a sow and the Bacillus strain is administered during gestation.

[0090] 54. The method of any one of clauses 27 to 53 wherein the feed composition is administered daily to the animal.

[0091] 55. The method of clause 27 wherein the animal is selected from the group consisting of a chicken, a pig, a horse, a pony, a cow, a turkey, a goat, a sheep, a quail, a pheasant, an ostrich, a duck, a fish, a crustacean, and combinations thereof.

[0092] 56. A commercial package comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF760421-435175(NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof.

[0093] 57. The commercial package of clause 56 further comprising a pharmaceutically acceptable carrier that stabilizes and enables the Bacillus strain to survive, reproduce, and grow in the digestive tract of the animal.

[0094] 58. The commercial package of clause 57, wherein the pharmaceutically acceptable earner comprises a natural or synthetic polymer, including for example, pectin, chitosan, guar gum, chondroitin sulfate, dextran, cyclodextrin, inulin, xanthan gum, amylose, alginates, shellac, locust bean gum and any combination thereof.

[0095] 59. A feed additive for an animal feed comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG- UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B- 68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof.

[0096] 60. An additive for the drinking water of an animal comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B- 68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof.

[0097] 61. An animal feed composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG- UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B- 68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain60421-435175MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof.

[0098] 62. The feed composition of clause 61 further comprising a nutrient formation wherein the nutrient formulation is coated or admixed with said Bacillus strains.

[0099] 63. The commercial package, feed additive, feed composition, or additive for the drinking water of the animal of any one of clauses 56 to 62 wherein the Bacillus strain causes an effect selected from the group consisting of preventing E. coli, Salmonella, Enterococcus and / or Clostridium disease, reducing E. coli, Salmonella, Enterococcus and / or Clostridium disease, maintaining the microbial balance of the animal, and combinations thereof.

[0100] 64. The commercial package, feed additive, feed composition, or additive for the drinking water of the animal of any one of clauses 56 to 63, wherein the Bacillus strain reduces E. coli, Salmonella, Enterococcus and / or Clostridium disease in the animal.

[0101] 65. The feed additive or additive for the drinking water of the animal of clause 59 or 60 in the form of a concentrate.

[0102] 66. The feed additive or additive for the drinking water of the animal of clause 59 or 60 in the form of a superconcentrate.

[0103] 67. The feed additive, feed composition, or additive for the drinking water of the animal of any one of clauses 59 to 66 in dry form.

[0104] 68. The feed composition of clause 67 in pelleted form.

[0105] 69. The commercial package of clause 56 wherein the strains are in a form selected from the group consisting of a powder, a liquid, and a pellet form.

[0106] 70. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 56 to 69 further comprising a earner for the Bacillus strains.

[0107] 71. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 70 wherein the carrier is selected from the group consisting of a bran, rice hulls, a salt, a dextrin, and combinations thereof.

[0108] 72. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 56 to 71 in a bag.

[0109] 73. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 72 wherein the bag is a plastic bag.60421-435175

[0110] 74. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 56 to 73 further comprising instructions for use of one or more of the Bacillus strains.

[0111] 75. The commercial package, feed additive, feed composition, or additive for the drinking water of the animal of any one of clauses 72 to 74 in a 20-pound bag.

[0112] 76. The commercial package, feed additive, feed composition, or additive for the drinking water of the animal of any one of clauses 72 to 74 in a 50-pound bag.

[0113] 77. The feed additive or additive for the drinking water of the animal of any one of clauses 60 to 67 in powder form.

[0114] 78. The feed additive or additive for the drinking water of the animal of any one of clauses 60 to 67 in liquid form.

[0115] 79. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 59 to 78 in a container for commercial use.

[0116] 80. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 79 wherein the container comprises plastic.

[0117] 81. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 79 wherein the container comprises paper.

[0118] 82. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 56 to 81 further comprising a binder, optionally wherein the binder is present at concentration of about 2-10% of the total weight of the commercial package, feed additive, additive for the drinking water of the animal, or feed composition.

[0119] 83. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 82 wherein the binder is selected from the group consisting of clay, yeast cell wall components, aluminum silicate, and glucan, or combinations thereof.

[0120] 84. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 56 to 83 comprising isolated Bacillus strain MDG-844 (NRRL No. B-68469), or a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), in combination with one or more of an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC2560421-435175(NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472).

[0121] 85. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 56 to 83 comprising isolated Bacillus stain MDG-UF7 (NRRL No. B-68470), or a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), in combination with one or more of an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-160 (NRRL No. B-68472), MDG-UC25 (NRRL No. B-68471), strain MDG-844 (NRRL No. B-68469) or a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471).

[0122] 86. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 56 to 74 comprising all of isolated Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG- UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472).

[0123] 87. A method of feeding an animal, the method comprising the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B- 68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B- 68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof.

[0124] 88. The method of clause 87 wherein the animal has a disease caused by E. coli, Salmonella, Enterococcus and / or Clostridium.

[0125] 89. The method of clause 87 wherein the animal has a disease caused by Salmonella.

[0126] 90. The method of clause 87 wherein the animal is selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal.

[0127] 91. The method of clause 90 wherein the poultry species is a broiler chicken.60421-435175

[0128] 92. The method of any one of clauses 87 to 91 wherein the E. coli, Salmonella, Enterococcus and / or Clostridium inhibition prevents E. coli, Salmonella, Enterococcus and / or Clostridium disease in the animal.

[0129] 93. The method of any one of clauses 87 to 91 wherein the E. coli, Salmonella, and / or Clostridium inhibition reduces E. coli, Salmonella, Enterococcus and / or Clostridium disease in the animal.

[0130] 94. The method of clause 90 wherein the animal is a porcine species and the porcine species is selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.

[0131] 95. The method of any one of clauses 87 to 94 wherein the Bacillus strain produces an enzyme selected from the group consisting of an a-galactosidase, a protease, a lipase, an amylase, a xylanase, a cellulase, and combinations thereof.

[0132] 96. The method of any one of clauses 87 to 95 further comprising the step of administering to the animal another bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[0133] 97. The method of any one of clauses 87 to 96 wherein the strain administered is Bacillus strain MDG-844 (NRRL No. B-68469).

[0134] 98. The method of any one of clauses 88 to 96 wherein the strain administered is Bacillus strain MDG-UF7 (NRRL No. B-68470).

[0135] 99. The method of any one of clauses 88 to 96 wherein the strain administered is Bacillus strain MDG-UC25 (NRRL No. B-68471).

[0136] 100. The method of any one of clauses 88 to 96 wherein the strain administered is Bacillus strain MDG-160 (NRRL No. B-68472).

[0137] 101. The method of any one of clauses 87 to 96 wherein Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472) are all administered.

[0138] 102. The method of any one of clauses 86 to 101 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 103CFU / gram of the feed composition to about 5.0 x 1012CFU / gram of the feed composition.

[0139] 103. The method of any one of clauses 87 to 101 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 103CFU / gram of the feed composition to about 1.0 x 10 CFU / gram of the feed composition.

[0140] 104. The method of any one of clauses 87 to 101 wherein the Bacillus strain is administered in the feed composition at a dose greater than about 7.0 x 104CFU / gram of the feed composition.60421-435175

[0141] 105. The method of any one of clauses 87 to 104 further comprising the step of administering an antibiotic to the animal.

[0142] 106. The method of any one of clauses 87 to 104 further comprising the step of administering to the animal an enzyme selected from the group consisting of a galactosidase, a protease, a lipase, an amylase, a hemicellulase, an arabinoxylanase, a xylanase, a cellulase, an NSPase, a phytase, and combinations thereof.

[0143] 107. The method of clause 106 wherein the enzyme is an NSPase or a phytase.

[0144] 108. The method of clause 90 wherein the companion animal is a canine species or a feline species.

[0145] 109. The method of clause 90 wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0146] 110. The method of clause 90 wherein the animal is a sow and the Bacillus strain is administered during gestation.

[0147] 111. The method of any one of clauses 87 to 110 wherein the feed composition is administered daily to the animal.

[0148] 112. The method of clause 90 wherein the animal is selected from the group consisting of a chicken, a pig, a horse, a pony, a cow, a turkey, a goat, a sheep, a quail, a pheasant, an ostrich, a duck, a fish, a crustacean, and combinations thereof.

[0149] 113. The commercial package of clause 56, where in the commercial package further comprises a fertilizer.

[0150] 114. A method of inhibiting pathogenic bacteria, optionally wherein the pathogenic bacteria are selected from E. coli, Salmonella, Enterococcus and / or Clostridium pathogens, said method comprising contacting said pathogenic bacteria with an effective amount of a composition comprising an isolated Bacillus strain, selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B- 68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B- 68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain MDG-160 (NRRL No. B-68472), and combinations thereof or a lysate, or a fermentation product of said Bacillus strain.

[0151] 115. The method of clause 114, wherein the pathogenic bacteria isSalmonella.60421-435175

[0152] 116. The method of clause 114 or 115, wherein the pathogenic bacteria are present on a surface of an article selected from the group consisting of animal bedding, animal bedding shavings, animal litter, manure, domesticated animal enclosures, crop plants, crop fields, produce, and meat products.

[0153] 117. The method of any one of clauses 114-116, wherein the surface is animal bedding.

[0154] 118. The method of any one of clauses 114-116, wherein the surface is animal bedding shavings.

[0155] 119. The method of any one of clauses 114-116, wherein the surface is animal litter.

[0156] 120. The method of any one of clauses 114-116, wherein the surface is manure.

[0157] 121. The method of any one of clauses 114-116, wherein the surface is domesticated animal enclosures.

[0158] 122. The method of any one of clauses 114-116, wherein the surface is crop plants.

[0159] 123. The method of any one of clauses 114-116, wherein the surface is crop fields.

[0160] 124. The method of any one of clauses 114-116, wherein the surface is produce.

[0161] 125. The method of any one of clauses 114-116, wherein the surface is meat products.

[0162] 126. The method of any one of clauses 110-125, wherein the composition is in the form of a powder or liquid.

[0163] 127. The method of any one of clauses 110-126, wherein the pathogenic bacteria is contacted with said composition via a spray, dip, or wash.

[0164] 128. The method of any one of clauses 110-127, wherein the strain contacting said surface is Bacillus strain MDG-844 (NRRL No. B -68469).

[0165] 129. The method of any one of clauses 110-127, wherein the strain contacting said surface is Bacillus strain MDG-UF7 (NRRL No. B-68470).

[0166] 130. The method of any one of clauses 110-127, wherein the strain contacting said surface is Bacillus strain MDG-UC25 (NRRL No. B-68471).

[0167] 131. The method of any one of clauses 110-127, wherein the strain contacting said surface is Bacillus strain MDG-160 (NRRL No. B-68472).60421-435175

[0168] 132. The method of any one of clauses 110-127, wherein Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472) are all applied to said surface either sequentially or combined in a single composition.

[0169] 133. The method of any one of clauses 110-132, wherein the Bacillus strain is applied at a concentration of about 1.0 x 103CFU / gram to about 5.0 x 1012CFU / gram.

[0170] 134. The method of any one of clauses 110-132, wherein the Bacillus strain is applied at a concentration of about 1.0 x 103CFU / gram to about 1.0 x 107CFU / gram.

[0171] 135. The method of any one of clauses 110-132, wherein the Bacillus strain is applied at a concentration greater than about 1.0 x 105CFU / gram.

[0172] 136. The method of any one of clauses 116-135, wherein the surface is a meat product.

[0173] 137. The method of any one of clauses 116-135, wherein the surface is harvested vegetables or fruit.

[0174] 138. The method of any one of clauses 116-135, wherein the surface is a prepared salad, ready-to-eat vegetables, or pre-cut vegetables and fruits.

[0175] 139. The method of any one of clauses 116-138, wherein the surface is contacted with a lysate, or a fermentation product of one or more of Bacillus strains selected from Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B- 68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain MDG-160 (NRRL No. B-68472).

[0176] 140. The method of any one of clauses 116-138, wherein the surface is contacted with a composition comprising a Bacillus strain selected from the group consisting of MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), and any combinations thereof.

[0177] In various embodiments, the animal to which a feed additive, a feed composition, or drinking water as described herein is administered can be selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal. In the embodiment where the animal is a companion animal, the companion animal can be, for example, a canine species or a feline species. In the embodiment where the animal is a porcine species, the porcine species60421-435175can be selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig. In various exemplary embodiments, the animal can be selected from the group consisting of a chicken (e.g., a broiler or a layer), a pig, a horse, a pony, a cow, a turkey, a goat, a sheep, a quail, a pheasant, an ostrich, a duck, a fish (e.g., a tilapia, a catfish, a flounder, or a salmon), a crustacean (e.g., a shrimp or a crab), and combinations thereof. In another embodiment, the feed additive, feed composition, or drinking water as described herein could be administered to a human.

[0178] In another embodiment, isolated Bacillus strains selected from the group consisting of MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG- UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof, can reduce contamination by detrimental E. coli, Salmonella, Enterococcus and / or Clostridium in meat products produced from the animal, or can be used in food processing to reduce detrimental effects of E. coli, Salmonella, Enterococcus and / or Clostridium in foods. In one embodiment the Bacillus compositions can be applied directly onto fresh vegetables, and fresh-ready to eat food such as deli salad, and used in washes for produce or in produce processing,

[0179] In various embodiments of the method, commercial package, feed additive, feed composition, or additive for drinking water described herein, the Bacillus strain can have an effect selected from the group consisting of maintaining microbial balance in the gut of the animal, preventing or reducing E. coli, Salmonella, Enterococcus and / or Clostridium disease in the animal, improving animal performance or health, maintaining gut health in the animal, reducing detrimental pathogens in the gut of the animal, odor reduction, reducing detrimental pathogens in the urine or feces of the animal, and preserving the growth of beneficial bacteria in the gut of the animal.

[0180] In any embodiments described herein, the Bacillus strains can be administered alone or in any combination, or can be in the form of any composition described herein so that the strains are alone or in any combination in the compositions described herein. The Bacillus strains described herein can also be used in combination with other microbial strains, including other Bacillus strains or Lactobacillus strains. Exemplary combinations include all of strains CM256 (NRRL No. B67706). CM857 (NRRL No. B67705), CM978 (NRRL No. used together60421-435175in combination with one of more of Bacillus strains MDG-844 (NRRL No. B-68469), MDG- UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472).

[0181] In one embodiment, a method of feeding an animal is provided. The method comprises the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof.

[0182] In another embodiment, a method of orally administering a beneficial Bacillus comprising composition to an animal is provided. The method comprises the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B- 68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B- 68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof, wherein the Bacillus strain causes Salmonella inhibition in the animal.

[0183] In one embodiment of the invention, an effective amount of the Bacillus strain can be administered to inhibit E. coli, Salmonella, Enterococcus and / or Clostridium in the animal. As used herein, “inhibit E. coli, Salmonella, Enterococcus and / or Clostridium” can mean reducing E. coli, Salmonella, Enterococcus and / or Clostridium disease, preventing E. coli, Salmonella, Enterococcus and / or Clostridium disease, maintaining the normal microbial balance in the animal, reducing the number of detrimental E. coli, Salmonella, Enterococcus and / or Clostridium in the animal, reducing the activity of E. coli, Salmonella, Enterococcus and / or Clostridium in the animal, or reducing the symptoms of E. coli, Salmonella, Enterococcus and / or Clostridium disease in the animal, or combinations thereof. By “effective amount” is meant an amount of the Bacillus strain (e.g., strain MDG-844 (NRRL60421-435175No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), or MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), capable of E. coli, Salmonella, Enterococcus and / or Clostridium inhibition or capable of controlling a detrimental effect of E. coli, Salmonella, Enterococcus and / or Clostridium, as described below, by any mechanism. In one illustrative embodiment, the animal has a disease caused by E. coli, Salmonella, Enterococcus and / or Clostridium.

[0184] In still another embodiment, a method of controlling a detrimental effect of E. coli, Salmonella, Enterococcus and / or Clostridium is provided. The method comprises the steps of administering to an animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B- 68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B- 68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof, and controlling the detrimental effect of E. coli, Salmonella, Enterococcus and / or Clostridium in the animal.

[0185] In embodiments described herein wherein the compositions of the present invention comprises an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof, are administered to an animal, the compositions are preferably administered to animals orally in a feed composition or in drinking water, but any other effective method of administration known to those skilled in the art may be utilized such as in a paste, a liquid drench, a top dress, or a capsule. In one illustrative embodiment, an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-84460421-435175(NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof, are provided in the form of an additive for addition to the drinking water of an animal. In another embodiment, the strains may be used as a litter treatment to control detrimental effects of E. coli, Salmonella, Enterococcus and / or Clostridium. In one embodiment the composition comprising the Bacillus strain MDG-844 (NRRL No. B-68469), Bacillus strain MDG-UF7 (NRRL No. B-68470), Bacillus strain MDG-UC25 (NRRL No. B-68471), and Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof is formulated as a liquid or a powder, wherein the liquid is sprayed, and the powder is dusted, directly onto the manure / feces, or a litter box contents, or shavings comprising manure / feces, or onto the bedding of an animal.

[0186] In another illustrative embodiment, an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B- 68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof, are provided in the form of a feed additive for addition to a feed composition. The feed composition may contain an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG- 844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B- 68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof, in a mixture with an animal feed blend, including any art- recognized animal feed blend or any animal feed blend described herein. As used herein, “feed composition” or “animal feed composition” means a feed composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-84460421-435175(NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof, in a mixture with an animal feed blend, and, optionally any other components that could be used in a feed composition, including other bacterial strains, such as other Bacillus strains or Lactobacillus strains. In one embodiment, the feed composition may be in the form of a ground meal.

[0187] Any animal feed blend, including those known in the art and those described herein, may be used in accordance with the methods and compositions described in this patent application, such as rapeseed meal, cottonseed meal, soybean meal, cornmeal, barley, wheat, silage, and haylage. In various embodiments, the animal feed blend can be supplemented with an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof, but other ingredients may optionally be added to the animal feed blend, including other bacterial strains, such as other Bacillus strains or Lactobacillus strains.

[0188] In various illustrative embodiments, optional ingredients of the animal feed blend include sugars and complex carbohydrates such as both water-soluble and waterinsoluble monosaccharides, di saccharides, and polysaccharides. Other optional ingredients include dried distillers grain solubles, fat (e.g., crude fat), phosphorous, sodium bicarbonate, limestone, salt, phytate, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, ash, fish oil, an oil derived from fish meal, raw seed (e.g., flaxseed), an antioxidant, and starch. In another embodiment, minerals may be added in the form of a mineral premix.

[0189] Optional amino acid ingredients that may be added to the animal feed blend are arginine, histidine, isoleucine, leucine, lysine, cysteine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine ethyl HC1, alanine, aspartic acid, sodium glutamate, glycine,60421-435175proline, serine, cysteine ethyl HC1, and analogs, and salts thereof. Vitamins that may be optionally added are thiamine HC1, riboflavin, pyridoxine HC1, niacin, niacinamide, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, and vitamins A, B, K, D, E, and the like. In another embodiment, vitamins may be added in the form of a vitamin premix. In yet another embodiment, protein ingredients may be added to the animal feed blend and include protein obtained from meat meal, bone meal, or fish meal, liquid or powdered egg, fish solubles, crude protein, and the like.

[0190] In another illustrative aspect, any medicament ingredients known in the art may be added to the animal feed blend or to an additive for the drinking water of the animal, such as antibiotics. In various embodiments, the antibiotic is selected from the group consisting of ampicillin, chloramphenicol, ciprofloxacin, clindamycin, tetracycline, chlortetracycline, Denagard™ (i.e., tiamulin), BMD™ (i.e., bacitracin methylene disalicylate), Carbadox™ (i.e., carbadox), Stafac™ (i.e., virginiamycin), erythromycin, levofloxacin, trimethoprim / sulfamethoxazole, trimethoprim, daptomycin, rifampicin, Tylan™ (i.e., tylosin), Pulmotil™ (i.e., tilmicosin), vancomycin, and combinations thereof. In another embodiment, the animal feed blend, the feed composition, the feed additive, or the additive for the drinking water of the animal may contain no antibiotics. In one embodiment an anti-coccidial, such as Salinomycin is added to the animal feed blend or to an additive for the drinking water of the animal, wherein the animal is a poultry species.

[0191] In another illustrative embodiment, one or more enzymes may be added to the animal feed blend. In various embodiments, the enzymes that may be added include a galactosidase, a phytase, a protease, a lipase, an amylase, a hemicellulase, an arabinoxylanase, a xylanase, a cellulase, an NSPase, combinations thereof, and any other enzyme that improves the effectiveness of the feed composition for E. coli, Clostridium, and / or Salmonella inhibition or controlling a detrimental effect of E. coli, Clostridium, and / or Salmonella. In yet another embodiment, yeast, fungi (e.g., Aspergillus or Trichodermd), or micronutrients may be added to the animal feed. Any of the ingredients described above that are suitable for addition to an additive for the drinking water of the animal may be added as a component of the additive for the drinking water of the animal as described herein.

[0192] In various illustrative embodiments, an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B- 68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain60421-435175MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof, can be administered in the feed composition at a dose of about 1.0 x 103CFU / gram of the feed composition to about 5.0 x 1012CFU / gram of the feed composition or at a dose of about 1.0 x 103CFU / gram of the feed composition to about 1.0 x 107CFU / gram of the feed composition. In other embodiments, the Bacillus strain is administered in the feed composition at a dose greater than about 1.0 x 103CFU / gram of the feed composition, at a dose greater than about 1.1 x 103CFU / gram of the feed composition, at a dose greater than about 1.25 x 103CFU / gram of the feed composition, at a dose greater than about 1.5 x 103CFU / gram of the feed composition, at a dose greater than about 1.75 x 103CFU / gram of the feed composition, at a dose greater than about 1.0 x 104CFU / gram of the feed composition, at a dose greater than about 2.0 x 104CFU / gram of the feed composition, at a dose greater than about 3.0 x 104CFU / gram of the feed composition, at a dose greater than about 4.0 x 104CFU / gram of the feed composition, at a dose greater than about 5.0 x 104CFU / gram of the feed composition, at a dose greater than about 6.0 x 104CFU / gram of the feed composition, at a dose greater than about 7.0 x 104CFU / gram of the feed composition, at a dose greater than about 8.0 x 104CFU / gram of the feed composition, at a dose greater than about 1.0 x 10' CFU / gram of the feed composition, at a dose greater than about 1.0 x 106CFU / gram of the feed composition, at a dose greater than about 1.0 x 10 CFU / gram of the feed composition, at a dose greater than about 1.0 x 10sCFU / gram of the feed composition, at a dose greater than about 1.0 x 10’ CFU / gram of the feed composition, at a dose greater than about 1.0 x IO10CFU / gram of the feed composition, at a dose greater than about 1.0 x 1011CFU / gram of the feed composition, or at a dose greater than about 1.0 x 1012CFU / gram of the feed composition. In yet another embodiment, the Bacillus strain is administered in the feed composition at a dose of about 1.23 x 105CFU per gram of the feed composition. In another embodiment, an isolated Bacillus strain selected from the group consisting of Bacillus strain 1.23 x 105CFU per gram of final feed, or any other bacterial strains added in addition to these Bacillus strains, can be administered in the feed composition at a dose of about 1.0 x IO2CFU / gram of the feed composition to about 5.0 x 1012CFU / gram of the feed composition or at a dose of about 1.0 x 104CFU / gram of the feed composition to about 1.0 x 106CFU / gram of the feed composition, or at a dose greater than about 1.0 x 105CFU / gram of the feed composition. In another embodiment, any of the dosages described herein can be in CFU / ml of drinking water in embodiments where the strains are administered in the drinking water of the animal.

[0193] In various embodiments, the isolated Bacillus strains described herein are selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-60421-435175UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B- 68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof. Bacillus strains MDG-844, MDG-UF7, MDG-UC25 and MDG-160 were deposited on November 14, 2024 at the Agricultural Research Service Culture Collection (NRRL), National Center for Agricultural Utilization Research, Agricultural Research Service, USDA, 1815 North University Street, Peoria, Illinois 61604-3999, and were given accession numbers B-68469, B-68470, B-68471 and B-68472, respectively. The deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Any and all restrictions imposed by Applicant on the availability to the public of the deposited material will be irrevocably removed upon the granting of the patent, and the deposits will be replaced if viable samples cannot be dispensed by the depository. The NRRL strain designations are B-68469, B-68470, B-68471 and B-68472, which are equivalent to Bacillus strain MDG-844, MDG-UF7, MDG- UC25 and MDG-160, respectively, as referred to in the application. Bacillus strain MDG-844, MDG-UF7, MDG-UC25 and MDG-160 are also referenced herein as Bacillus strain 844, UF7, UC25 and 160, respectively, and represent the same species. Any of these strains can be administered alone or in combination in the form of a feed composition (e.g., a complete feed comprising an animal feed blend) or drinking water for an animal. In one embodiment, multiple strains are administered in combination in a single composition. In another embodiment, multiple strains are administered in combination in separate compositions.

[0194] In another embodiment, one or more of the Bacillus strains, described in the preceding paragraphs, including isolated Bacillus strains selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG- UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof, can be administered to the animal along with another bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof. In yet another embodiment, one or more60421-435175of isolated Bacillus strains selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof, can be administered to the animal along with any other bacterial strain effective to inhibit or control detrimental effects of E. coli, Salmonella, Enterococcus and / or Clostridium in the animal.

[0195] As used herein “a strain having all of the identifying characteristics of’ Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), or MDG-160 (NRRL No. B-68472), can be a mutant strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), MDG- UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), or MDG-160 (NRRL No. B- 68472), (e.g., a DNA fingerprint based on DNA analysis that corresponds to the DNA fingerprint of Bacillus strain Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), or MDG-160 (NRRL No. B- 68472), enzyme activities that correspond to Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), or MDG-160 (NRRL No. B-68472), antimicrobial activity that corresponds to Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), or MDG-160 (NRRL No. B-68472), , antibiotic sensitivity and tolerance profiles that correspond to Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG- UC25 (NRRL No. B-68471), or MDG-160 (NRRL No. B-68472), or combinations of these identifying characteristics). In alternate embodiments, the mutation can be a natural mutation, or a genetically engineered mutation. In another embodiment, “a strain having all of the identifying characteristics of’ Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), or MDG-160 (NRRL No. B- 68472) can be a strain, for example, produced by isolating one or more plasmids from Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG- UC25 (NRRL No. B-68471), or MDG-160 (NRRL No. B-68472) and introducing the one or more plasmids into another bacterium, such as another Bacillus strain, as long as the one or more plasmids contain DNA that provides the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No.60421-435175B-68471), or MDG-160 (NRRL No. B-68472) (e.g., a DNA fingerprint based on DNA analysis that corresponds to the DNA fingerpri nt of Bacillus strain MDG-844 (NRRL No. B- 68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), or MDG-160 (NRRL No. B-68472).

[0196] The feed composition or drinking water comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG- UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B- 68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof, may be administered to the animal for any time period that is effective to inhibit E. coli, Salmonella, Enterococcus and / or Clostridium or control a detrimental effect of E. coli, Salmonella, Enterococcus and / or Clostridium in the animal, or combinations thereof. For example, in one embodiment the feed composition or drinking water may be provided to the animal daily. In an alternate embodiment, the feed composition or drinking water may be administered to the animal during lactation and / or during gestation. The time periods for administration of the feed composition or drinking water described above are non-limiting examples and it should be appreciated that any time period or administration schedule determined to be effective to inhibit E. coli, Salmonella, Enterococcus and / or Clostridium or control a detrimental effect of E. coli, Salmonella, Enterococcus and / or Clostridium, or combinations thereof, may be used.

[0197] In these embodiments, “controlling a detrimental effect of E. coli, Salmonella, Enterococcus and / or Clostridium" means at least one of the following: reducing E. coli, Salmonella, Enterococcus and / or Clostridium disease, preventing E. coli, Salmonella, Enterococcus and / or Clostridium disease, maintaining the normal microbial balance in the animal, reducing the number of detrimental E. coli, Salmonella, Enterococcus and / or Clostridium in the animal, reducing the activity of E. coli, Salmonella, Enterococcus and / or Clostridium in the animal, or reducing the symptoms of E. coli, Salmonella, Enterococcus and / or Clostridium, disease in the animal, or combinations thereof. By “effective amount” is meant an amount of the Bacillus strain capable of controlling a detrimental effect of E. coli, Salmonella, Enterococcus and / or Clostridium, as described above, by any mechanism.

[0198] In additional embodiments of the invention, compositions comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-60421-43517568469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B -68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof, are provided.

[0199] In yet another illustrative aspect of the invention, an animal feed composition is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof, optionally in combination with nutrients and standard feed components.

[0200] In one embodiment, the feed additive for addition to an animal feed blend to produce a complete feed composition can be mixed with the animal feed blend, for example, with an automated micro-nutrient delivery system, or, for example, by hand-weighing and addition to achieve any of the doses of an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B- 68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B- 68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof, described herein, for administration to the animal in the form of a complete feed composition. The mixing can also be done by any other suitable method known in the art for combining direct-fed microbials with an animal feed blend to obtain a uniform mixture. In various embodiments, the mixing can be done for any suitable time period (e.g., about 1 to about 4 minutes). In the embodiment where an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG- 844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B- 68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics60421-435175of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof, is in the form of an additive for the drinking water of the animal, the isolated Bacillus strain can be in the form of, for example, a powder, a liquid, or pellets, and can be mixed with the drinking water using any suitable method known in the art to achieve any of the doses of the Bacillus strains described herein, for administration to the animal in the drinking water of the animal.

[0201] The isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof, can also be fed directly to the animal orally (e.g., by oral insertion) in the form of a powder, a liquid, or a pellet.

[0202] In one illustrative aspect, the feed additive, additive for the drinking water of the animal, or the feed composition can be in the form of a commercial package. In another illustrative embodiment, the feed additive or additive for the drinking water of the animal, or the isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof, in the commercial package can be in the form of a concentrate (e.g., about 1 x 10sto about 5 x 109CFU / g) or a superconcentrate (e.g., about 1 x 1010to about 5 x 1012CFU / g). In another embodiment, the feed additive, feed composition, or additive for the drinking water of the animal, comprises the isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No.60421-435175B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof, formulated as a dry form (e.g., a powder), a pelleted form, a liquid form, in the form of a top-dressing, or in the form of a gel, or any other suitable form.

[0203] In yet another embodiment, the strains that are in the form of a commercial package can be, for example, in a dry form (e.g., a powder or freeze-dried form), in a pelleted form, or in a liquid form.

[0204] In another illustrative embodiment, the commercial package, feed additive, additive for the drinking water of the animal, or feed composition can further comprise a carrier for the isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof. The carrier can be selected from the group consisting of a bran, rice hulls, a salt, mineral oil, a dextrin (e.g., maltodextrin), whey, sugar, sucrose, limestone, yeast culture, dried starch, sodium silico aluminate, silicon dioxide, polypropylene glycol, polysorbate 80, vegetable oil, and combinations thereof. In another embodiment, the carrier can be any suitable carrier known in the art for a direct -fed microbial. In another embodiment, the commercial package, feed additive, additive for the drinking water of the animal, or feed composition can further comprise a binder such as clay, yeast cell wall components, aluminum silicate, glucan, or other known binders. In another embodiment, the commercial package, feed additive, additive for the drinking water of the animal, or feed composition can further comprise inorganic / organic binders, essential oils, and / or organic acids.

[0205] In yet other embodiments, the commercial package, feed additive, additive for the drinking water of the animal, or feed composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG- UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B- 68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain60421-435175MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B-68472), and combinations thereof, is in a container for commercial use. In various embodiments the container can be, for example, a bag (e.g., a 20-pound bag, a 50-pound bag, a 2-ounce bag, a 1 -pound bag, or a 1 -kilogram bag), a pouch, a drum, a bottle, or a box. In illustrative aspects, the container for the commercial package, feed additive, additive for the drinking water of the animal, or feed composition disclosed herein can comprise plastic, metal, foil, paper, fiber, or cardboard (e.g., a plastic pail, a paper bag, a foil bag, a fiber drum, etc.). The commercial package, feed additive, additive for the drinking water of the animal, or feed composition can further comprise instructions for use of one or more of the Bacillus strains.

[0206] In another illustrative embodiment, the supernatants from the culture of any of the isolated Bacillus strains described herein selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain, MDG-160 (NRRL No. B- 68472), and combinations thereof, can be used in the same methods and compositions and for the same purpose as described herein for the whole cell preparations of the strains.

[0207] The following examples are for illustrative purposes only. The examples are non-limiting and are not intended to limit the invention in any way.EXAMPLESExample 1: Inhibition of serotyped Salmonella isolates in antimicrobial susceptibility assays.

[0208] Experimental Design and Methods;

[0209] Salmonella isolates were cultured from a variety of sources including gastrointestinal tissues and environmental samples from pre-harvest poultry, ruminant, swine, and the American Type Culture Collection (ATTC) using xylose lysine deoxycholate (XLD, Neogen) or xylose-lysine-tergitol 4 (XLT4) agar (Becton Dickenson). Cultures were incubated at 37 °C for 24 hours and further propagation of presumptive positive colonies was performed in tryptic soy broth (TSB, Becton Dickenson) under the same growth conditions. Isolates were confirmed as Salmonella via a species-specific qPCR. Salmonella isolates were screened for susceptibility to Bacillus strains 160 (NRRL No. B-68472), Bacillus strain 84460421-435175(NRRL No. B-68469), Bacillus strain UC25 (NRRL No. B-68471), and Bacillus strain UF7 (NRRL No. B-68470).

[0210] The screening was performed via one of two methods: a broth antimicrobial susceptibility assay or an agar antimicrobial susceptibility assay.

[0211] The screening was performed in a broth antimicrobial susceptibility assay, using a 1% (v / v) inoculation of a 24-hour pathogen culture into tryptic soy broth containing >5% (v / v) of a cell-free Bacillus fermentate from each Bacillus strain. Assays were incubated for 16 hours at 37 °C under aerobic growth conditions. Optical density (ODeoo) was measured, and percentage of inhibition was calculated in comparison to each pathogen isolate’ s positive control grown in tryptic soy broth without Bacillus fermentate. Growth inhibition detected for each Salmonella isolate and Bacillus strain combination was scored as weak (<10% inhibition,-), moderate (10-25% inhibition, + / -), strong (25-50% inhibition, +), and very strong (>50% inhibition, ++).

[0212] The screening was also performed in an agar antimicrobial susceptibility assay where isolates were tested against a panel of Bacillus strains using the cross-streak method on brain heart infusion agar (Becton Dickenson). For the agar antimicrobial susceptibility assay, brain heart infusion agar plates were streaked across the midline with a 1 pl calibrated loop of Bacillus culture and incubated for 16 hours at 32 °C. Salmonella isolates were inoculated onto cross-streak plates with a 1 pl calibrated loop perpendicular to the Bacillus streak. After incubating at 37 °C for 16 hours, Salmonella growth was examined, and a zone of inhibition was measured in mm from the edge of the Bacillus streak. Growth inhibition detected for each Salmonella isolate and Bacillus strain combination was scored as weak (<1 mm inhibition, -), moderate (2-3 mm inhibition, + / -), strong (4-5 mm inhibition, +), or very strong (>5 mm inhibition, ++).

[0213] Results:

[0214] The in vitro growth of Salmonella isolates were inhibited by one or more strains of Bacillus strain 160 (NRRL No. B-68472), Bacillus strain 844 (NRRL No. B-68469), Bacillus strain UC25 (NRRL No. B-68471), and Bacillus strain UF7 (NRRL No. B-68470). Growth inhibition of Salmonella from these environments provides an example mechanism by which these specific Bacillus strains can reduce Salmonella in poultry, ruminant, and swine.

[0215] Table 1. In vitro inhibitory activity of specific Bacillus strains against serotyped Salmonella isolates from a variety of sources including gastrointestinal tissues, litter, and environmental samples from pre-harvest poultry, ruminant, swine, and the American Type Culture Collection (ATTC). Growth inhibition is classified as weak (<10% or <1 mm60421-435175inhibition,-), moderate (10-25% or 2-3 mm inhibition, + / -), strong (25-50% or 4-5 mm inhibition, +), and very strong (>50% or >5 mm inhibition, ++).60421-435175Table 160421-435175Example 2: Inhibition of non-serotyped Salmonella isolates in a broth antimicrobial susceptibility assay.

[0216] Experimental Design and Methods;

[0217] Salmonella isolates were cultured from a variety of sources including gastrointestinal tissues and environmental samples from pre- and post-harvest poultry, ruminant, swine, and the American Type Culture Collection (ATTC) using xylose lysine deoxycholate (XLD, Neogen) or xylose-lysine-tergitol 4 (XLT4) agar (Becton Dickenson). Cultures were incubated at 37 °C for 24 hours and further propagation of presumptive positive colonies was performed in tryptic soy broth (TSB, Becton Dickenson) under the same growth conditions. Isolates were confirmed as Salmonella via a species-specific qPCR. A total of 893 Salmonella isolates were screened for susceptibility to fermentate from Bacillus strains 16060421-435175(NRRL No. B-68472), Bacillus strain 844 (NRRL No.68469), Bacillus strain UC25 (NRRL No. 68471), and Bacillus strain UF7 (NRRL No. 68470). The screening was performed in a broth antimicrobial susceptibility assay, using a 1% (v / v) inoculation of a 24-hour pathogen culture into tryptic soy broth containing >5% (v / v) of a cell-free Bacillus fermentate from each Bacillus strain. Assays were incubated for 16 hours at 37 °C under aerobic growth conditions. Optical density (ODeoo) was measured, and percentage of inhibition was calculated in comparison to each pathogen isolate’ s positive control grown in tryptic soy broth without Bacillus fermentate. Growth inhibition detected for each Salmonella isolate and Bacillus strain combination was scored as weak (<10% inhibition,-), moderate (10-25% inhibition, + / -), strong (25-50% inhibition, +), and very strong (>50% inhibition, ++).

[0218] Results;

[0219] The in vitro growth of Salmonella isolates were inhibited strongly by the presence of fermentate from one or more strains of Bacillus strains 160 (NRRL No. B-68472), Bacillus strain 844 (NRRL No. B-68469), Bacillus strain UC25 (NRRL No. B-68471), and Bacillus strain UF7 (NRRL No. B-68470). Growth inhibition of Salmonella from these environments provides an example mechanism by which these specific Bacillus strains can reduce Salmonella in poultry, ruminant, and swine.

[0220] Table 2. In vitro average inhibitory activity of specific Bacillus strains against non-serotyped Salmonella isolates from a variety of sources including gastrointestinal tissues, litter, and environmental samples from pre- and post-harvest poultry, ruminant, swine, and the American Type Culture Collection (ATTC). Growth inhibition is classified as an average percent inhibition as weak (<10% inhibition,-), moderate (10-25% inhibition, + / -), strong (25- 50% inhibition, +), and very strong (>50% inhibition, ++).60421-435175Table 2

[0221] Example 3: Inhibition of other pathogenic bacteria species in a broth antimicrobial susceptibility assay (E coli).

[0222] Experimental Design and Methods:

[0223] E. coli isolates were cultured from a variety of environments including gastrointestinal tissues and environmental samples from pre-harvest poultry using sorbitol- MacConkey agar (SMAC, Becton Dickenson). Cultures were incubated at 37°C for 24 hours and further propagation of presumptive positive colonies was performed in tryptic soy broth (TSB, Becton Dickenson) under the same growth conditions. Isolates were confirmed as E. coli via a species-specific qPCR. A total of 161 E. coli isolates were screened for susceptibility to fermentate from Bacillus strains 160 (NRRL No.), Bacillus strain 844 (NRRL No.), Bacillus strain UC25 (NRRL No.), and Bacillus strain UF7 (NRRL No.). The screening was performed in a broth antimicrobial susceptibility assay, using a 1% (v / v) inoculation of a 24-hour pathogen culture into tryptic soy broth containing >5% (v / v) of a cell-free Bacillus fermentate from each Bacillus strain. Assays were incubated for 16 hours at 37 °C under aerobic growth conditions. Optical density (ODeoo) was measured, and percentage of inhibition was calculated in comparison to each pathogen isolate’ s positive control grown in tryptic soy broth without Bacillus fermentate. Growth inhibition detected for each E. coli60421-435175isolate and Bacillus strain combination was scored as weak (<10% inhibition,-), moderate (10-25% inhibition, + / -), strong (25-50% inhibition, +), and very strong (>50% inhibition, ++)•

[0224] Results:

[0225] The in vitro growth of E. coli isolates were inhibited by the presence of fermentate from one or more strains of Bacillus strain 160 (NRRL No. B-68472), Bacillus strain 844 (NRRL No. B-68469), Bacillus strain UC25 (NRRL No. B-68471), and Bacillus strain UF7 (NRRL No. B-68470). Growth inhibition of E. coli from these environments provide an example mechanism by which these specific Bacillus strains can reduce common coinfection pathogens like E. coli in poultry.

[0226] Table 3. In vitro average inhibitory activity of specific Bacillus strains against E. coli isolates from a variety of environments including gastrointestinal tissues and litter from pre-harvest poultry. Growth inhibition is classified as an average percent inhibition as weak (<10% inhibition,-), moderate (10-25% inhibition, + / -), strong (25-50% inhibition, +), and very strong (>50% inhibition, ++).Table 3Example 4: Inhibition of other pathogenic bacteria species in a broth antimicrobial susceptibility assay (Clostridium perfringens).

[0227] Experimental Design and Methods:

[0228] Clostridium perfringens isolates were cultured from a variety of environments including gastrointestinal tissues and litter from pre -harvest poultry using tryptose sulphite cycloserine (TSC) agar and Perfringens TSC supplement (Thermo Scientihc™Oxoid™). Cultures were incubated anaerobically at 37°C for 24 hours and further propagation of presumptive positive colonies was performed in brain heart infusion broth (BHI, Becton Dickenson) under the same growth conditions. Isolates were confirmed as C. perfringens via a species-specific qPCR. A total of 49 C. perfringens isolates were screened for susceptibility to fermentate from Bacillus strains 160 (NRRL No. B-68472), Bacillus strain 844 (NRRL No. B-68469), Bacillus strain UC25 (NRRL No. B-68471), and Bacillus strain UF7 (NRRL No. B-68470). The screening was performed in a broth antimicrobial susceptibility assay, using a 1% (v / v) inoculation of a 24-hour anaerobic pathogen culture into brain heart infusion broth60421-435175containing >5% (v / v) of a cell-free Bacillus ferment ate from each Bacillus strain. Assays were incubated for 24 hours at 37 °C under anerobic growth conditions. Optical density (OD60O) was measured, and percentage of inhibition was calculated in comparison to each pathogen isolate’ s positive control grown in brain heart infusion without Bacillus fermentate. Growth inhibition detected for each C. perfringens isolate and Bacillus strain combination was scored as weak (<10% inhibition,-), moderate (10-25% inhibition, + / -), strong (25-50% inhibition, +), and very strong (>50% inhibition, ++).

[0229] Results:

[0230] The in vitro growth of C. perfringens isolates were inhibited strongly by the presence of fermentate from one or more strains of Bacillus 160 (NRRL No. B-68472), Bacillus strain 844 (NRRL No. B-68469), Bacillus strain UC25 (NRRL No. B-68471), and Bacillus strain UF7 (NRRL No. B-68470). Growth inhibition of C. perfringens from these environments provide an example mechanism by which these specific Bacillus strains can reduce common coinfection pathogens like C. perfringens in poultry.

[0231] Table 4. In vitro average inhibitory activity of specific Bacillus strains against C. perfringens isolates from a variety of environments including gastrointestinal tissues and litter from pre -harvest poultry. Growth inhibition is classified as an average percent inhibition as weak (<10% inhibition,-), moderate (10-25% inhibition, + / -), strong (25-50% inhibition, +), and very strong (>50% inhibition, ++).Table 4

[0232] Example 5: Inhibition of other pathogenic bacteria species (Enterococcus).

[0233] Experimental Design and Methods:

[0234] Enterococcus species, cecorum and faecalis, were cultured from a variety of environments including gastrointestinal tissues and litter from pre-harvest poultry using Columbia Nalidixic Acid agar (CNA) with 5% sheep blood (Hardy Diagnostics) and BBL Enterococcosel agar (Becton Dickenson) respectively. Cultures were incubated at 37°C for 48 hours anaerobically for E. cecorum and aerobically for E. faecalis. Further propagation of presumptive positive colonies was performed in brain heart infusion broth (BHI, Becton60421-435175Dickenson) incubated at 37°C for 24 hours anaerobically for E. cecorum and aerobically for E. faecalis. Isolates were confirmed as E. cecorum or E. faecalis via a species-specific qPCR. A total of 63 E. faecalis and 6 E. cecorum isolates were screened for susceptibility to Bacillus strains 160 (NRRL No. B-68472), Bacillus strain 844 (NRRL No. B-68469), Bacillus strain UC25 (NRRL No. B-68471), and Bacillus strain UF7 (NRRL No. B-68470). The screening was performed in an agar antimicrobial susceptibility assay where isolates were tested against a panel of Bacillus strains using the cross-streak method on brain heart infusion agar (Becton Dickenson). For the agar antimicrobial susceptibility assay, brain heart infusion agar plates were streaked across the midline with a 1 pl calibrated loop of Bacillus culture and incubated for 16 hours at 32°C. Enterococcus isolates were inoculated onto cross-streak plates with a 1 pl calibrated loop perpendicular to the Bacillus streak. After incubating at 37°C for 16 hours, Enterococcus growth was examined, and a zone of inhibition was measured in mm from the edge of the Bacillus streak. Growth inhibition detected for each Enterococcus isolate and Bacillus strain combination was scored as weak (<lmm inhibition, -), moderate (2-3mm inhibition, + / -), strong (4-5mm inhibition, +), or very strong (>5mm inhibition, ++).

[0235] Results;

[0236] The in vitro growth of Enterococcus isolates were inhibited strongly by one or more strains of Bacillus strains 160 (NRRL No. B-68472), Bacillus strain 844 (NRRL No. B- 68469), Bacillus strain UC25 (NRRL No. B-68471), and Bacillus strain UF7 (NRRL No. B- 68470). Growth inhibition of Enterococcus from these environments provide an example mechanism by which these specific Bacillus strains can reduce common coinfection pathogens like Enterococcus in poultry.

[0237] Table 5. In vitro average inhibitory activity of specific Bacillus strains against Enterococcus species isolates from a variety of environments including gastrointestinal tissues and litter from pre-harvest poultry. Growth inhibition is classified as an average percent inhibition as weak (<lmm inhibition, -), moderate (2-3mm inhibition, + / -), strong (4- 5mm inhibition, +), or very strong (>5mm inhibition, ++).60421-435175Table 5

[0238] Example 6: In vivo studies of the efficacy of Bacillus strains on broiler chickens during a Salmonella challenge.

[0239] Experimental Design and Methods;

[0240] In a controlled research facility, the efficacy of a Bacillus strain combination in reducing Salmonella quantity in birds’ ceca was determined in a concrete floor pen study using broiler chickens with appropriate depth of clean wood shavings. This trial used Ross 708 birds with 270 birds per treatment (10 replicates of 27 birds per pen) in a randomized block design. Negative control birds received no challenge and no Bacillus, positive control birds received a Salmonella challenge and no Bacillus, while treated birds received a Salmonella challenge and Bacillus strain 844 (NRRL No. B-68469) and Bacillus strain 160 (NRRL No. B-68472). Treated birds received Bacillus strain 160 (NRRL No. B-68472) and Bacillus 844 (NRRL No. B-68469) in the feed at 0.5 lb / U.S. ton, designed to deliver 1.23 x 105CFU per gram of final feed. Birds received their respective treatments throughout the entire 21 -day trial period. Challenged birds without the administration of Bacillus were compared to birds supplemented with Bacillus strain 160 (NRRL No. B-68472) and Bacillus strain 844 (NRRL No. B-68469). Food and water were provided ad libitum throughout the study. On day 7, all of the birds in both positive control and Bacillus treatments were directly infected with Salmonella Enteritidis by oral gavage using 1.0 mL of 1.0 x IO9CFU / mL Salmonella enterica subsp. enterica Enteritidis culture harboring a nalidixic acid resistance marker. On day 21, 4 randomly selected birds per pen were euthanized and cecal lobes removed to determine Salmonella Enteritidis infection. Salmonella Enteritidis was enumerated by serial dilution and plating on XLT4 agar containing nalidixic acid.

[0241] Results;

[0242] Bacillus strain 160 (NRRL No. B-68472) and Bacillus strain 844 (NRRL No. B- 68469) reduced the Salmonella gene quantity in the ceca of the birds on day 21, 14 days post inoculation with Salmonella.60421-435175

[0243] Table 6. Salmonella recovered in the ceca at d21 of 40 broiler chickens per treatment, 14 days post inoculation with as Salmonella challenge. Salmonella quantity is an average measured in logio CFU / g.Table 6

[0244] Example 7: In vivo effect of Bacillus in a Caenorhabditis elegans gut colonization assay.

[0245] Experimental Design and Methods: Synchronized populations of Caenorhabditis elegans hermaphrodites were fed one Bacillus strain for three days from the first larval stage (LI) through adulthood. Subsequently, worms were transferred to Salmonella enterica subsp. enterica Enteritidis for three days. Twelve individual worms were homogenized per treatment and plated on xylose lysine deoxycholate (XLD, Neogen) to quantify Salmonella CFUs per worm to determine Salmonella gut colonization by treatment. The following strains were fed individually, Bacillus strains strain 160 (NRRL No. B-68472), Bacillus strain 844 (NRRL No. B-68469), and Bacillus strain UC25 (NRRL No. B-68471).

[0246] Results:

[0247] Prophylactic exposure to Bacillus strain 844 (NRRL No. B-68469) reduced Salmonella gut colonization compared to worms fed the control E. coli OP50 and other Bacillus treatments. See Fig. 1.

[0248] Example 8: Salmonella biofilm inhibition by Bacillus.60421-435175

[0249] Objective: The objective of the biofilm inhibition assay was to determine the effect of Bacillus strain 160 (NRRL No. B-68472), Bacillus strain 844 (NRRL No. B-68469), and Bacillus strain UF7 (NRRL No. B-68470) on the inhibition of a Salmonella biofilm.

[0250] Experimental Design and Methods:

[0251] A Salmonella Enteritidis isolate was cultured in tryptic soy broth (TSB, Becton Dickenson) and incubated shaking at 200 RPM for 16 hours at 37°C, then standardized to an optical density (ODeoo) of 0.6 and inoculated at 1% (v / v) into tryptic soy broth containing >5% (v / v) of a cell-free fermentate from Bacillus strain 160 (NRRL No. B-68472), Bacillus strain 844 (NRRL No. B-68469), ox Bacillus strain UF7 (NRRL No. B-68470) individually. Cultures were incubated statically in microtiter plates (Nunc MicroWell Plates, Thermo Fisher) for 24 hours at 37°C. After incubation, media was carefully removed, and wells were washed twice with PBS (Gibco). Biofilms were then fixed by drying plates upside down at 55°C for 30 minutes, stained with 0.1% crystal violet for 15 minutes, rinsed twice with PBS and dried for >4 hours. The stain was then solubilized with 30% acetic acid for 20-30 minutes. Final optical density (OD595) values were used to quantify the crystal violet stain, and ergo the degree of biofilm formation, biofilm inhibition was compared across treatments.

[0252] Results:

[0253] Exposure to cell-free fermentates from Bacillus strain 844 (NRRL No. B-68469) and Bacillus strain UF7 (NRRL No. B-68470) inhibited biofilm growth of a poultry origin Salmonella isolate (See Fig 2).

[0254] Example 9: Inhibition of Salmonella Enteritidis in an in vitro challenge model.

[0255] Objective: The objective of the in vitro challenge study was to determine the effect of Bacillus strains 160 (NRRL No. B-68472), Bacillus strain 844 (NRRL No. B-68469), Bacillus strain UC25 (NRRL No. B-68471), and Bacillus strain UF7 (NRRL No. B-68470) on the inhibition of a Salmonella growth in a test tube by measuring the quantity of the highly conserved gene, invA, across all Salmonella serovars via qPCR.

[0256] Experimental Design and Methods:

[0257] A poultry-origin Salmonella Enteritidis challenge isolate, utilized in the “In vivo studies of the efficacy of Bacillus strains on broiler chickens during a Salmonella challenge”60421-435175was cultured in tryptic soy broth (TSB, Becton Dickenson) for 16 hours at 37° C, then standardized to an optical density (ODeoo) of 0.4, representing approximately IxlO8CFU / ml, followed by serial dilution in sterile peptone diluent to reach a concentration of approximately IxlO3CFU / ml. Bacillus strains were cultured in tryptic soy broth at 130 RPM for 16 hours at 32 °C, then standardized to an optical density (ODgoo) of 0.5 using sterile cell free-fermentate from the same strain to obtain approximately 1.3xlO6CFU / ml. One ml of standardized Salmonella Enterilidis was incubated with one ml of individual standardized Bacillus inoculum as prepared for each strain and 8 mis of tryptic soy broth. Controls in this experiment include standardized Salmonella Enteritidis alone and a negative tryptic soy broth control. All treatment tubes were incubated at 130 RPM for 4 hours at 37° C, followed by cooling all samples to halt Salmonella growth. DNA was extracted (QIAmp PowerFecal Pro DNA Kit, Qiagen), and qPCR was completed to quantify the Salmonella invasion gene, invA, by treatment. Differences by treatment were determined by a one-way ANOVA using post-hoc pairwise comparisons with Bonferroni correction. Superscripts display significance p<0.001.

[0258] Results;

[0259] Addition of Bacillus strain 844 (NRRL No. B-68469), Bacillus strain UC25 (NRRL No. B-68471), or Bacillus strain UF7 (NRRL No. B-68470) reduced Salmonella invA gene quantities significantly by more than 1 log compared to the control (p<0.001). Data for this experiment is presented in Fig. 3.

[0260] Example 10; Effect of Bacillus on Salmonella adhesion and invasion of IPEC- J2 cells.

[0261] Objective; The objective of this experiment was to determine the effect of Bacillus strains 160 (NRRL No. B-68472) and 844 (NRRL No. B-68469) on Salmonella adhesion and invasion of non-transformed porcine intestinal epithelial cells originally derived from the jejunum of a neonatal unsuckled piglet and designated as IPEC-J2 cells.

[0262] Experimental Design and Methods;

[0263] Swine intestinal epithelial cells of the IPEC-J2 line were grown to 100% confluency in a flask in DMEM / F12 broth (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12, Gibco) with growth and antibiotic additives then seeded into 12-well plates to form a monolayer at 32°C and 5% CO2 (growth was approximately 4 days to reach confluence). Once confluent, media was replaced with DMEM / F12 without additives and phenol red (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12, no phenol red, Gibco), to allow60421-435175for cell differentiation, and cells were incubated for 16-24 hours at 32°C and 5% CO2. A poultry-origin Salmonella Enteritidis challenge isolate, utilized in the “In vivo studies of the efficacy of Bacillus strains on broiler chickens during a Salmonella challenge” was cultured in tryptic soy broth (TSB, Becton Dickenson) at 130 RPM for 16 hours at 37°C, then Salmonella was pelleted and rinsed with PBS (Gibco) and standardized in DMEM / F12 without additives and phenol red to an optical density (OD60o) of 0.4. Bacillus strains were cultured in tryptic soy broth at 130 RPM for 16 hours at 32 °C, then standardized to an optical density (ODeco) of 0.5 followed by dilution in sterile cell free-fermentate from the same strain. 250 pl of Bacillus inoculum was mixed with 2.25 ml of standardized Salmonella inoculum and incubated for 1 hour at 37°C and 5% CO2 to allow time for cellular interaction between the Bacillus and the Salmonella. Once the Salmonella and Bacillus were incubated by treatment, the IPEC-J2 cells were rinsed with PBS (Gibco), and the Salmonella (approximately 1x10sCFU / ml) mA Bacillus blends (approximately lxl()5CFU / ml) were applied to the monolayers and incubated for 1 hour at 37°C and 5% CO2to allow for Salmonella attachment and invasion of the IPEC-J2 cells. IPEC-J2 cells were lysed with 0.1% triton X-100; lysate was collected to capture both attached and intracellular (invaded) Salmonella. Collected IPEC-J2 lysate was serially diluted in sterile peptone diluent, plated on xylose lysine deoxycholate (XLD, Neogen), incubated at 37°C for 16-24 hours, and enumerated to quantify Salmonella adherence and invasion. Differences by treatment were determined by a one-way ANOVA using post -hoc pairwise comparisons with Bonferroni correction. Superscripts display significance p<0.001.

[0264] Results:

[0265] Addition of Bacillus strain 844 (NRRL No. B-68469) significantly reduced the attached or invaded Salmonella of the swine intestinal epithelial cells of the IPEC-J2 line (p<0.001). See data presented in Fig. 4

Claims

1. 60421-435175WHAT IS CLAIMED IS:

1. A feed additive, for an animal feed or for the drinking water of an animal, said feed additive comprising an isolated Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain MDG-160 (NRRL No. B-68472), and combinations thereof.

2. An animal feed composition comprising a nutrient formulation, optionally wherein the nutrient formulation comprises one or more compounds selected from the group consisting of lipids, protein, fiber, carbohydrate, minerals and vitamins; andan isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain MDG-160 (NRRL No. B-68472), and combinations thereof.

3. A commercial package comprising an isolated Bacillus strain, selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain MDG-160 (NRRL No. B-68472), and combinations thereof, or a cell lysate or fermentate of said Bacillus strain.

4. The commercial package, feed additive, or feed composition of any one of claims 1 to 3, wherein the Bacillus strains are present in a concentrated form of about 1 x 108to about 5 x 10' CFU / g. or a superconcentrate (e.g., about 1 x IO10to about 5 x 1012CFU / g).60421-4351755. The commercial package, feed additive, or feed composition of any one of claims 1 to 3, wherein the Bacillus strains are present in a supperconcentrated form of about 1 x IO10to about 5 x 1012CFU / g.

6. The commercial package, feed additive, or feed composition of any one of claims 1 to 5 in dry form.

7. The commercial package, feed additive, or feed composition of any one of claims 1 to 6 in pelleted form, optionally in combination with a binder, optionally at 2-10% of the total weight of the pellets.

8. The commercial package, feed additive, or feed composition of claim 7, wherein the binder is selected from the group consisting of clay, yeast cell wall components, aluminum silicate, and glucan, or combinations thereof.

9. The commercial package, feed additive, or feed composition of any one of claims 1 to 6, wherein the strains are in a form selected from the group consisting of a powder, a liquid, and a pellet form.

10. The commercial package, feed additive, or feed composition of any one of claims 1 to 6, wherein the strains are in a pellet form.

11. The commercial package, feed additive, or feed composition of any one of claims 1 to 6, further comprising a carrier for the Bacillus strains.

12. The commercial package, feed additive, or feed composition of any one of claims 1 to 6, wherein the carrier is selected from the group consisting of a bran, rice hulls, a dextrin, and combinations thereof.

13. The commercial package, feed additive, or feed composition of claim 11, wherein the carrier is a pharmaceutically acceptable carrier selected from the group consisting of gelatin, cellulose, and starch, or any combination thereof, wherein the Bacillus strain is stabilized by the pharmaceutically acceptable carrier such that the Bacillus strain is able to survive, reproduce, and grow in the digestive tract of the animal.

14. The commercial package, feed additive, or feed composition of any one of claims 1 to 6 in powder form.

15. The commercial package, feed additive, or feed composition of any one of claims 1 to 6 in liquid form.

16. The commercial package, feed additive, or feed composition of any one of claims 1 to 6 further comprising a binder.

17. The commercial package, feed additive, or feed composition of claim 16, wherein the binder is selected from the group consisting of clay, yeast cell wall components, aluminum silicate, and glucan, or combinations thereof.60421-43517518. A method of controlling a detrimental effect in an animal resulting from E. coli, Salmonella, Enterococcus and / or Clostridium, the method comprising the steps of administering to said animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain MDG-160 (NRRL No. B-68472), and combinations thereof, and controlling the detrimental effect of E. coli, Salmonella, and / or Clostridium in the animal.

19. The method of claim 20 wherein the animal has a disease caused by E. coli, Salmonella, Enterococcus and / or Clostridium.

20. The method of claim 18 or 19 wherein the animal is selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal.

21. The method of claim 20 wherein the animal is a poultry species, optionally a broiler chicken.

22. The method of claim 20 wherein the animal is a porcine species and the porcine species is selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.

23. The method of any one of claims 18 to 22 wherein the E. coli, Salmonella, Enterococcus and / or Clostridium inhibition prevents or reduces the incidence of E. coli, Salmonella, Enterococcus and / or Clostridium disease in the animal.

24. The method of any one of claims 18 to 23 wherein the strain administered is Bacillus strain MDG-844 (NRRL No. B-68469).

25. The method of any one of claims 18 to 23 wherein the strain administered is Bacillus strain MDG-UF7 (NRRL No. B-68470).

26. The method of any one of claims 18 to 23 wherein the strain administered is Bacillus strain MDG-UC25 (NRRL No. B-68471).

27. The method of any one of claims 18 to 23 wherein the strain administered is Bacillus strain MDG-160 (NRRL No. B-68472).

28. The method of any one of claims 18 to 23 wherein the additive comprises two or more Bacillus strains selected from the group consisting of Bacillus strains MDG-844 (NRRL60421-435175No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472)29. The method of any one of claims 18 to 23 wherein the additive comprises Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472).

30. The method of any one of claims 18 to 29 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10 CFU / gram of the feed composition to about 5.0 x 1012CFU / gram of the feed composition.

31. The method of any one of claims 18 to 29 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 103CFU / gram of the feed composition to about 1.0 x 107CFU / gram of the feed composition.

32. The method of any one of claims 18 to 29 wherein the Bacillus strain is administered in the feed composition at a dose greater than about 1.0 x 10 CFU / gram of the feed composition.

33. The method of any one of claims 18 to 29 further comprising the step of administering an antibiotic to the animal.

34. The method of any one of claims 18 to 29 wherein the feed composition is administered daily to the animal.

35. A method of inhibiting pathogenic bacteria, optionally wherein the pathogenic bacteria are selected from E. coli, Salmonella, Enterococcus and / or Clostridium pathogens, on a surface, said method comprising contacting said pathogenic bacteria with a composition comprising an isolated Bacillus strain, selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain MDG-160 (NRRL No. B-68472), and combinations thereof or a lysate, or a fermentation product of one or more of said Bacillus strains.

36. The method of claim 35, wherein the pathogenic bacteria is a Salmonella strain.

37. The method of claim 35 or 36, wherein the surface is on an article selected from the group consisting of animal bedding, animal bedding shavings, animal litter, manure, domesticated animal enclosures, crop plants, crop fields, produce and meat products.60421-43517538. The method of any one of claims 35-37, wherein the composition is in the form of a powder or liquid.

39. The method of claim 35, wherein the pathogenic bacteria is contacted with said composition via a spray, dip, or wash.

40. A method of feeding an animal, the method comprising the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472), a strain having all of the identifying characteristics of Bacillus strain MDG-844 (NRRL No. B-68469), a strain having all of the identifying characteristics of Bacillus strain MDG-UF7 (NRRL No. B-68470), a strain having all of the identifying characteristics of Bacillus strain MDG-UC25 (NRRL No. B-68471), a strain having all of the identifying characteristics of Bacillus strain MDG-160 (NRRL No. B-68472), and combinations thereof, wherein the Bacillus strain causes E. coli, Salmonella, Enterococcus and / or Clostridium inhibition in the animal.

41. The method of claim 40 wherein the animal has a disease caused by E. coli, Salmonella, Enterococcus and / or Clostridium.

42. The method of claim 40 or 41 wherein the animal is selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal.

43. The method of claim 42 wherein the animal is a poultry species, optionally a broiler chicken.

44. The method of claim 40 wherein said additive comprises an isolated Bacillus strain selected from the group consisting of Bacillus strain strain MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472), and any combination thereof.

45. The method of any one of claims 40 to 44 wherein the E. coli, Salmonella, Enterococcus and / or Clostridium inhibition prevents or reduces the incidence of E. coli, Salmonella, Enterococcus and / or Clostridium disease in the animal.

46. The method of claim 42 wherein the animal is a porcine species and the porcine species is selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.

47. The method of claim 4 wherein the Bacillus strain causes Salmonella inhibition in the animal.60421-43517548. The method of any one of claims 40 to 47 wherein the strain administered is Bacillus strain MDG-844 (NRRL No. B-68469).

49. The method of any one of claims 40 to 47 wherein the strain administered is Bacillus strain MDG-UF7 (NRRL No. B-68470).

50. The method of any one of claims 40 to 47 wherein the strain administered is Bacillus strain MDG-UC25 (NRRL No. B-68471).

51. The method of any one of claims 40 to 47 wherein the strain administered is Bacillus strain MDG-160 (NRRL No. B -68472).

52. The method of any one of claims 40 to 47 wherein Bacillus strains MDG-844 (NRRL No. B-68469), MDG-UF7 (NRRL No. B-68470), MDG-UC25 (NRRL No. B-68471), and MDG-160 (NRRL No. B-68472) are all administered.

53. The method of any one of claims 40 to 52 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 103CFU / gram of the feed composition to about 5.0 x 1012CFU / gram of the feed composition.

54. The method of any one of claims 40 to 52 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 103CFU / gram of the feed composition to about 1.0 x 107CFU / gram of the feed composition.

55. The method of any one of claims 40 to 52 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 104CFU / gram of the feed composition to about 1.0 x 106CFU / gram of the feed composition.

56. The method of any one of claims 40 to 55 further comprising the step of administering an antibiotic to the animal.

57. The method of any one of claims 40 to 56 wherein the feed composition is administered daily to the animal.

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